The prevalence of heart failure (HF) is increasing in the developed world and the cost of providing medical care for an expanding HF population imposes an increasingly heavy burden on healthcare systems throughout the world. Most commonly, HF is associated with impaired left ventricular (LV) systolic function. However, at least half of all patients with typical symptoms of congestive HF have a normal or slightly reduced left ventricular ejection fraction (LVEF) (>50%). The predominant cause of heart failure with preserved ejection fraction (HFpEF) is diastolic heart failure (DHF). Heart failure (HF) with preserved ejection fraction (HFpEF) is predominantly caused by hypertension, is often preceded by asymptomatic left ventricular diastolic dysfunction (ALVDD) and has few defined therapies. The predominant aetiological cause of DHF is myocardial fibrosis as a result of long standing hypertension and metabolic abnormalities associated with diabetes and obesity. The rising prevalence of metabolic disease due to the obesity and diabetic epidemics means that DHF is a major public health problem. DHF, similar to systolic HF has a five-year mortality rate of 65%. In many of these patients, diastolic dysfunction caused by hypertensive heart disease (HHD) is implicated as a major contributor, if not a primary cause. Furthermore, the prevalence of asymptomatic diastolic dysfunction in the community is significant with approximately 25-30% of individuals >45 years of age being affected. There are no proven, life-saving therapies for treating DHF. Many of the well-established drug therapies for systolic heart failure have been directed at DHF without success. The diagnosis of DHF can present a challenge in routine clinical practice. The major limitation in the diagnosis of DHF is the identification of diastolic dysfunction (DD), which at present is predominantly reliant on Doppler echocardiographic studies. Echocardiography has been used for many years to provide structural correlates to the clinical picture of HF. It can also measure multiple clinically important parameters of cardiac function, including hemodynamic status and LVEF, volumes and mass. The pathophysiology of DHF includes delayed relaxation, impaired LV filling and/or increased stiffness. These conditions result in an upward displacement of the diastolic pressure-volume relationship with increased end-diastolic, left atrial and pulmo-capillary wedge pressure leading to symptoms of pulmonary congestion. Diagnosis of DHF requires three conditions; (1) presence of signs or symptoms of HF; (2) presence of normal or slightly reduced LVEF (>50%) and (3) presence of increased diastolic filling pressure. Data indicate that the underlying pathophysiology in diastolic dysfunction and DHF is related to myocardial interstitial disease. Collagen is a stable protein and its balanced turnover is estimated to be 80-120 days. Alteration of collagen turnover by various mechanisms can lead to adverse accumulation of collagen in the myocardial interstitium leading to fibrosis, increased tissue stiffness, reduced myocardial compliance and impaired diastolic function. The successful neurohumoral-based approach to pharmacotherapy in HF with systolic dysfunction has not resulted in similarly impressive results in HFpEF, implicating additional pathophysiological signals. Changes in the extracellular matrix (ECM), known as myocardial remodeling, are central abnormalities in many patients with HFpEF and are characterized by inflammation, increased ECM turnover and myocardial fibrosis. Key mediators of inflammation are pro-inflammatory cytokines including interleukins (IL) (IL-1β, IL-6, IL-8) and tumor necrosis factor (TNF)α. Key regulators of the turnover of collagen and extracellular matrix (ECM) in the myocardium are the matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). MMPs in particular have been found to play an important role in both inflammation and fibrosis. MMPs also contribute to collagen degradation and remodeling of the ECM after myocardial infarction. ECM turnover is regulated by matrix metalloproteinases (MMPs), especially the “gelatinases”, MMP-2 and MMP-9, and their tissue inhibitors (TIMPs). MMP-2 and MMP-9 knockout models are associated with reduced aortic elastin degradation and protection from pressure overload hypertrophy, fibrosis and dysfunction. In the clinic, independent associations between ALVDD and HFpEF have been identified with markers of inflammation, fibrosis and MMP-9. During ischemic cardiomyopathy, neutrophil proteinase activates latent myocardial MMP, which can degrade the ECM. If unchecked by TIMPs, the ECM continuously degrades, leading to ventricular dilatation and diastolic dysfunction. Despite the emerging awareness of the potential role of collagen metabolism in the pathogenesis of diastolic HF there are as yet no effective therapies for this form of HF. Pharmacological modulation of MMPs may present an opportunity. However, all MMP synthetic inhibitors developed to date have either been ineffective or demonstrated dose- and duration-dependent drug-related side-effects, most which were musculoskeletal-related. Despite some promising animal studies of MMP inhibitors showing attenuation of cardiovascular remodeling in chronic pressure-overload models, the approach of direct inhibition of MMP enzymes has proven too toxic or ineffective in the clinic. An alternative approach in cardiovascular disease would inhibit production and/or secretion of inducible myocardial MMP-9. As well as classic inflammatory diseases such as rheumatoid arthritis, hay fever, periodontitis, inflammation plays an important role in the development and progression of diabetes and a variety of cardiovascular conditions, most notably coronary atherosclerosis and congestive heart failure. The term “Diabetic cardiomyopathy” was coined 4 decades ago and describes a “silent, stiffening” of the heart tissue which can lead to heart failure. There are no symptoms until heart failure occurs. It is present in half of people with diabetes and is more prevalent than well-recognised “silent pumping problem” which has good treatment available. This silent stiffening of the heart is linked to overweight, diabetes, high blood pressure and there are no specific therapies. Over the past 20 years, basic and human research has shown that enzymes in the heart called matrix metallproteinases or MMPs are involved in the stiffening process. They also affect large and small blood vessels and cause eye and kidney damage in diabetes. For example, in patients with diabetic retinopathy, increased MMP-9 activity was observed in retinal microvessels and MMP-9 knockout was protective (Kowluru et al, Abrogation of MMP-9 Gene Protects Against the Development of Retinopathy in Diabetic Mice by Preventing Mitochondrial Damage. Diabetes. 2011 Sep. 20 [Epub ahead of print]). Increased urinary excretion of MMP-9 in patients supports a role for MMP-9 dysregulation in diabetic renal dysfunction (Thrailkill et al., Endocrine. 2010 April; 37(2):336-43). Aortic and coronary arteries of diabetic patients taken at autopsy had higher expression of MMP-9 compared to non-diabetics and were correlated with HbA1c as well as apoptosis (Ishibashi et al., J Atheroscler Thromb. 2010 Jun. 30; 17(6):578-89). Elevated MMP-9 has also been associated with arterial stiffness in patients with diabetes (Chung et al., Cardiovasc Res. 2009 Dec. 1; 84(3):494-504). Furthermore, human genetic polymorphisms associated with MMP-9 elevation support a role for this enzyme in the pathophysiology of vascular disease. The 1562C>T single nucleotide polymorphism (SNP), which affects the promoter region of MMP-9 gene and increases circulating levels of MMP-9, is significantly associated with vascular disease in type 2 diabetes mellitus (Wang et al., Biochem Biophys Res Commun. 2010 Jan. 1; 391(1):113-7). In age and sex matched controls, patients with type 2 diabetes without and with microangiopathy, T allele frequencies were 11.9%, 13.1% and 24.4% respectively (p<0.05). Similarly, in a cohort of asymptomatic hypertensive patients, the 1562C>1 polymorphism is associated with increased T allele frequency, higher plasma MMP-9 and evidence of increased hypertension and vascular stiffness, measured by pulse wave velocity (Zhou et al. J Hum Hypertens. 2007 November; 21(11):861-7). Inflammation is also involved in the development and progression of some cancers (e.g., gallbladder carcinoma). Inflammation is mediated by a complex interplay of mediators such as IL-1 beta, IL-4 and IL-8. IL-1 beta induces COX-2, which causes brain levels of prostaglandin (PG)E2 to rise, thus activating the thermoregulatory center for fever production. In the periphery, IL-1 beta activates IL-1 receptors on the endothelium, resulting in expression of adhesion molecules and chemokines, which facilitate the emigration of neutrophils into the tissue spaces. IL-1 is pro-inflammatory and has been implicated in various pro-inflammatory diseases such as coronary atherosclerosis and congestive heart failure as well as diabetes where recent studies from animals, in-vitro cultures and clinical trials provide evidence that support a causative role for IL-1β as the primary agonist in the loss of beta-cell mass in type 2 diabetes. IL-4 is a TH2 type anti-inflammatory and profibrosis cytokine that stimulates and amplifies the inflammatory response by activation of the synthesis of types I and II collagen by fibroblasts and the promotion of the progression of fibrosis. IL-4 also inhibits the proinflammatory response of TNF-α, IL-1 and IL-6. IL-4 stimulates inflammatory responses, activates collagen synthesis, promotes fibrosis progression, and inhibits the production of inflammatory cytokines. The patients with CHF had higher IL-4 and PIIINP values than the controls. Comparison of the IL-4 values between the patients and controls showed a significantly greater difference in the CHF patients (12 [12] vs 4 [3] pg/mL; P<0.0001). Recent studies have shown that pro-inflammatory cytokines play a significant contributory role in the pathogenesis of acute heart failure. The purpose of this study was to determine whether the serum IL-8 concentration in patients with acute myocardial infarction (AMI), who were undergoing percutaneous coronary intervention (PCI) was related to the subsequent presence or absence of heart failure. A study by Dominguez-Rodriguez 2006, included 50 patients who underwent successful PCI. During their subsequent stay in the coronary care unit, their maximum degree of heart failure was recorded. Serum levels of IL-8 in patients more severe symptoms (Killip class >I) were significantly higher than those of with less severe symptoms (Killip class I) (P<0.001). By multivariante analysis a higher level of IL-8 was a significant predictor of heart failure after PCI. Similarly in HF, the presence of the metabolic syndrome which puts patients at higher risk, plasma levels of IL-8 (p<0.05) were significantly higher in HF patients with MetS than those without MetS.
Tetracyclines, commonly known for their broad-spectrum antimicrobial properties, have been characterized as pleiotropic immunomodulatory agents. In human studies, sub-antimicrobial doses of the tetracycline, doxycycline, have exerted potentially beneficial effects on inflammation that could promote plaque stability in an effort to prevent acute coronary syndrome, as doxycycline therapy has been shown to lead to a powerful reduction of aneurysmal wall neutrophil and cytotoxic T-cell count; two cell types considered crucial for the process of aneurysm formation. Attempts have been made to attenuate MMP expression to inhibit aortic abdominal aneurysm formation using doxycycline, thereby reducing the need for surgery. Doxycycline has been shown to inhibit secretion of MMP-2 and MMP-9 and is the only drug currently licensed for human use that relies on MMP inhibition. It is currently under evaluation in ALVDD and HF patients in our group for its effects on inflammation, MMPs, myocardial structure and function using cardiac MRI [EudraCT number: 2010-021664-16]. However, in several animal and human studies, the efficacy of MMP inhibition with doxycycline has been questioned. This may reflect non-specific inhibition of the wider MMP family with high doses and/or chronic therapy, involving inhibition of both constitutive and inducible enzymes. It prompted our group to create analogues of doxycycline that target over-expression of inducible MMP-9 rather than direct enzyme inhibition as a more effective and safer approach. Evidence is emerging that members of the MMP and/or A disintegrin and metalloproteinase (ADAM) family can serve not only as potential markers for diagnosis and prognosis, early detection, and risk assessment, but also as indicators of tumor recurrence, metastatic spread, and response to primary and adjuvant therapy for breast cancer. MMP-9 levels in tumor tissue as well as serum, plasma, and urine are significantly elevated in patients with breast cancer. Recently, efforts have focused on the use of MMPs and ADAMs as potential biomarkers of early breast cancer. Studies indicate that urinary MMP-9 and ADAM12, in addition to being predictive markers for breast cancer, may also prove useful as noninvasive breast cancer risk assessment tools. Several independent studies have used circulating MMP-9 activity to predict metastatic spread of disease as well as to monitor patient response to primary and adjuvant therapy and to evaluate outcome. High levels of serum MMP-9 and TIMP-1 are associated with increased incidence of lymph node metastasis and decreased relapse-free and overall survival rates. MMPs may also be useful in predicting therapeutic efficacy. Plasma MMP-9 levels decrease after the surgical removal of primary breast tumors and a progressive decrease in plasma MMP-9 was observed in patients who responded well to adjuvant therapy. Importantly, in all patients who suffered a relapse of disease there was a gradual increase of plasma MMP-9 activity 1 to 8 months before the clinical diagnosis of recurrence. Serum and tissue levels of MMP-9 are significantly higher in patients with pancreatic ductal adenocarcinoma than in patients with chronic pancreatitis and healthy controls. Active MMP-2 levels are upregulated in the pancreatic juice of patients with cancer (100%) as compared with patients with chronic pancreatitis (2%) or normal controls (0%). Several studies have reported that plasma and/or serum levels of MMP-9 and TIMP-1 are elevated in patients with stage III or IV lung cancer when compared with those in patients with nonmalignant lung diseases. Urinary MMP-2 and MMP-9 levels correlate with presence of bladder cancer as well as stage and grade of disease. Several MMP species have been reported in urine from patients with primary tumors in the bladder and prostate including MMP-2, MMP-9, MMP-9/neutrophil gelatinase-associated lipocalin complex and MMP-9 dimer. Each urinary MMP species was detected at significantly higher rates in urine from patients with cancer as compared with controls. The difference in detection of MMP species in the urine of the two types of cancers studied may serve as a tumor-specific fingerprint that can indicate both the presence of a tumor as well as its location. Increased levels of MMP-9 and MMP-2 in urine correlate with increased expression of these proteases in bladder tumor tissue as well. Urinary MMP-9 levels when combined with telomerase analysis of exfoliated cells from voided urine could also increase the sensitivity of cytology, a commonly used method for bladder cancer detection and monitoring. MMP-2 and MMP-9 have been studied as potential prognostic biomarkers of colorectal cancer. Enhanced MMP-9 staining in primary tumors was found to be an independent marker of poor prognosis in a study with T3-T4 node-negative patients. Plasma MMP-2 and MMP-9 levels were significantly elevated in patients with colorectal cancer and those with adenomatous polyps, and significant reduction in both were observed after tumor resections, suggesting their potential as markers for therapeutic efficacy. These MMPs may not be prognostic markers for tumor recurrence, however, since plasma proMMP-2 and -9 activities did not correlate with disease relapse after surgery. Tutton and colleagues investigated whether plasma MMP-2 and MMP-9 levels could be used as a surrogate for tumour expression in colorectal cancer patients and they found significant correlations between plasma levels and tumor pre- and post-op. MMP-2, -9, and -14 are among the most studied MMPs as biomarkers for ovarian cancer. MMP-9 activity in tissue extracts was significantly increased in advanced ovarian cancers (International Federation of Gynecology and Obstetrics stage III) compared with benign tumors and was found to be an independent prognosticator of poor survival. In another study of invasive epithelial ovarian cancer, high stromal expressions of MMP-9 and -14 were significantly correlated with cancer progression and were independent prognostic markers. Tissue MMPs have also been shown to distinguish different histotypes of ovarian cancer, which is a significant finding given that different histotypes have different prognoses. A recent study showed that more than 90% of clear-cell carcinomas expressed moderate to high levels of MMP-2 or MMP-14, compared with 30% to 55% of the other ovarian cancer histotypes (serous, endometroid, and mucinous), whereas MMP-9 was expressed more widely in other histotypes. Importantly, the cellular source of MMPs must be considered when evaluating MMPs as ovarian cancer biomarkers. For example, strong MMP-9 levels in cancer cells were associated with longer survival whereas strong stromal MMP-9 was associated with shorter survival, suggesting a dual role for MMP-9 during ovarian cancer progression. MMP-2, -9, -15, and -26 expression in tissue or serum have been positively correlated with Gleason score in prostate cancer. Among these MMPs, the activities of plasma MMP-2 and -9 increased significantly in metastatic prostate cancer. Analysis of MMP-2 and -9 levels in radical prostatectomy specimens revealed these two as significant predictors of cancer recurrence. These two enzymes may also be markers of therapeutic efficacy, since both the levels and activities of plasma MMP-2 and -9 decreased significantly in metastatic patients after therapy. In addition, increased urinary MMP-9 activity has been shown to distinguish between prostate and other types of cancer (e.g. bladder cancer). MMPs can also be combined with other markers to increase their predictive capability. For example, the mRNA ratio of gelatinases to E-cadherin in biopsy samples independently predicted prostate cancer stage. Elevated tissue levels of MMP-2 and MMP-9 have been reported in aggressive brain tumors. Both latent and activated forms of MMP-2 and MMP-9 have been detected in the cerebrospinal fluid of patients with brain tumors. In studies of primary glial tumors and other central nervous system tumors, we have recently shown that detection of MMP-2, MMP-9, MMP-9/neutrophil gelatinase-associated lipocalin complex, and/or vascular endothelial growth factor in the urine predicted disease status and therapeutic efficiency of patients with brain cancer. Importantly, these studies showed that the upregulation of MMP-2 and -9 in the source tumor tissue was also reflected in CSF as well as in urine of these patients. Tumor cells overexpress proteases and/or induce expression of these enzymes in neighboring stromal cells in order to degrade the basement membrane and invade the surrounding tissue. Several MMPs have been implicated in the ECM degradation associated with tumor growth and angiogenesis. This proteolytic activity is also required for a cancer cell to invade a nearby blood vessel (intravasation) and then extravasate at a distant location and invade the distant tissue in order to seed a new metastatic site. MMPs have been shown to promote angiogenesis through their release of angiogenic factors stored in the ECM such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF; 3). Stroma-derived MMP-9 can facilitate the liberation of ECM-sequestered VEGF during tumor angiogenesis. MMPs play complex and sometimes conflicting roles in regulating angiogenesis. Remodeling of the ECM during angiogenesis is accomplished largely through the activity of MMPs. Angiogenic mitogens, such as bFGF and VEGF, can stimulate the production of MMPs by capillary endothelial cells. Studies have also demonstrated that MMPs are involved in the angiogenic switch, one of the earliest stages of tumor growth and progression. It has been shown that MMP-9 can be a regulator of the angiogenic switch in a pancreatic tumor model, further confirming the pro-angiogenic role of MMPs. These findings strongly suggest that MMP activity is critical, not only to the initiation of angiogenesis, but to the maintenance of the growing vascular bed, which in turn supports tumor growth and metastasis. MMP activity can, however, result in the production of negative regulators of angiogenesis as well. ECM degradation products display unique biologic properties that can trigger a variety of cellular signals. MMPs have also been implicated in the epithelial to mesenchymal transition (EMT), a hallmark of cancer progression to metastasis. Activation of growth factors and cleavage of adhesion molecules are some of the proposed mechanisms underlying MMP-induced EMT. Recent studies point to an emerging role for MMPs in modulating aspects of immunity and inflammation during tumorigenesis. A variety of cytokines, cytokine receptors, and chemokines have been found to undergo MMP-mediated cleavage. In breast cancer, MMP-9 expression is upregulated in tumor-associated stromal cells including neutrophils, macrophages, and lymphocytes and may play a role in tumor-associated inflammation. Several members of the MMP and ADAM family can regulate cellular proliferation by modulating the bioavailability of growth factors or cell-surface receptors. Ligands for several growth factor receptors are processed by MMP/ADAM family members as well. There are known clinical benefits of MMP inhibition in cancer management (for example Neovastat (AstraZeneca) is currently under evaluation in phase II renal cell carcinoma). However, most MMP inhibitors are too toxic for use in the clinic and adverse effects of MMP inhibitors (e.g. musculoskeletal adverse effects) limit their use. Furthermore, there may be problems with potent, broad spectrum, MMP inhibition. For example, there are some data suggesting that tumour progression is inversely proportional to MMP-3. Accordingly, it is not known if MMP-3 sparing or MMP-3 inhibiting effects are preferable. Recent developments in anti-cancer agents targeting the matrix metalloproteinases have been reviewed (Li, et al., Recent Patents on Anti-Cancer Drug Discovery 2010, 5: 109-141) and show that MMP inhibitors are classified into three main pharmacologic categories: Collagen peptidomimetics, non-peptidomimetics and tetracycline derivatives. Collagen peptidomimetics can be further subdivided into hydroxamates, carboxylates, aminocarboxylates, sulfhydryls, phosphoric acid derivatives. Most MMP inhibitors in clinical development are hydroxamate derivatives, e.g. batimastat and marimastat, illomastat. The lead compounds have been largely unsuccessful because of toxicity and or lack of efficacy. For example, Batimastat can only be administered intraperitoneally and intrapleurally and further development has been suspended. In the case of Marimastat, no benefit over placebo was seen in patients with breast and lung cancer. Severe musculoskeletal pain occurred in 18% of patients and quality of life worsened with marimastat therapy. Development of this drug has also been discontinued. Several members of the non-peptidomimetics class of compounds are undergoing evaluation in Phase III studies in cancer patients. However, the majority are no longer in development because of an adverse efficacy/toxicity profile (including AG3340/Prinomastat (Agouron), BMS-275291 (Bristol-Myers-Squibb), CGS27023A/MMI270 (Novartis), Bay12-9566/Tanomastat (Bayer Inc). Neovastat/AE-941 (Aetherna Zentaris) has MMP-2, MMP-9 and VEGF inhibitory properties and is being evaluated as a potential treatment of renal carcinoma and Phase II clinical trials are underway. Some tetracycline derivatives, such as doxycycline and COL-3 have been evaluated in preclinical cancer models and G31 have entered early clinical trials in patients. Doxycycline has been shown to substantially reduce the tumor burden from breast cancer metastasis in nude mice. It exerts diverse inhibitor effects on MMP production and activity, inhibits tumor cell proliferation. However, it accumulates at high concentrations in bone, and can therefore be used for the treatment of bone metastasis. Inhibition of mitochondrial protein synthesis by doxycycline has significant anti-tumor effects in several tumor systems. Continuous doxycycline treatment combined with intermittent administration of adriamycin or 1-beta-D-arabinofuranosyl cytosine on the growth of rat leukemia resulted in the delay of tumor relapse. Treatment with zoledronic acid in combination with doxycycline may be very beneficial for breast cancer patients at risk for osteolytic bone metastasis, according to the fact that administration of a combination of zoledronic acid and doxycycline resulted in a 74% decrease in total tumor burden compared to untreated mice. In addition, doxycycline significantly enhances the tumor regression activity of cyclophosphamide, a widely used chemotherapeutic drug in neoplasias, on xenograft mice model bearing MCF-7 cells, suggesting that such combination chemotherapeutic regimen may lead to additional improvements in treatment of breast cancer. In vivo, the inhibitory effects of doxycycline on breast cancer tumor matastasis formation was potentiated by the addition of batimastat, confirming that targeting MMPs through multiple distinct pathways may improve treatment efficacy. However, in a Phase I evaluation of cancer patients, oral doses of 400 mg administered twice a day resulted in dose-limiting toxicity that consisted of fatigue, confusion, nausea, and vomiting. At the maximum tolerated dose of 300 mg twice a day, mean through plasma concentrations were comparable to those associated with antiangiogenic effect in vivo.
Nitric oxide is a gaseous molecule that is unsuitable for oral administration. However, there are several pharmacologically relevant nitric oxide-donor groups than are known to release nitric oxide in response to conditions found in the human body after administration. Exemplary nitric-donor groups are described in “Nitric Oxide Donors: For Pharmaceutical and Biological Applications”; Peng George Wang, Tingwei Bill Cai, Naoyuki Taniguchi, Wiley (2005), the contents of which are incorporated herein by reference. The effects of nitric oxide on MMPs are complex. Nitric oxide has been reported to possess inhibitory effects on MMP-9 by destabilization of MMP-9 RNA and through effects on MMP-9 activating cytokines, secondary messengers and transcription factors (AP-1). In contrast higher concentrations of nitric oxide have been shown to cause MMP activation through S-nitrosylation of an inhibitory cysteine on the prodomain.
Abbreviations: ALVDD=Asymptomatic left ventricular diastolic dysfunction, AUC=Area under the curve, cGMP=Cyclic guanosine monophosphate, DMSO=Dimethyl supfoxide, DNA=Deoxyribonucleic acid, ECM=Extracellular matrix, FCS=Fetal calf serum, HCF=Human ventricular cardiac fibroblasts, HF=Heart failure, HFpEF=Heart failure with preserved ejection fraction, IF=Interferon, iNOS=Inducible nitric oxide synthase, IQR=Interquartile range, MCP=Monocyte chemotactic protein, MMP=Matrix metalloproteinase, MRI=Magnetic resonance imaging, mRNA=messenger ribonucleic acid, NHP=Non-human primate, NO=Nitric oxide, PBMC=Peripheral blood mononuclear cells, PCR=Polymerase chain reaction, RAAS=Renin-angiotensin-aldosterone system, RNA=Ribonucleic acid, SEM=Standard error of the mean, TIMP=Tissue inhibitor of matrix metalloproteinase, TNFα=Tumor necrosis factor alpha.